Screening biofilm eradication activity of ethanol extracts from foodstuffs: potent biofilm eradication activity of glabridin, a major flavonoid from licorice (Glycyrrhiza glabra), alone and in combination with ɛ-poly-L-lysine.

The ethanol extracts of 155 different foodstuffs containing medicinal plants were investigated for their biofilm eradication activities against pathogenic bacteria. A combined method of a colorimetric microbial viability assay based on reduction of a tetrazolium salt (WST-8) and a biofilm formation technique on the 96-pins of a microtiter plate lid was used to screen the biofilm eradication activities of foodstuffs. The ethanol extracts of licorice (Glycyrrhiza glabra) showed potent biofilm eradication activities against Streptococcus mutans, Staphylococcus aureus, and Porphyromonas gingivalis. Among the antimicrobial constituents in licorice, glabridin had the most potent eradication activities against microbial biofilms. The minimum biofilm eradication concentration of glabridin was 25-50 µg/ml. Furthermore, the combination of glabridin with ɛ-poly-L-lysine, a food additive, could result in broad biofilm eradication activities towards a wide variety of bacteria associated with infection, including Escherichia coli and Pseudomonas aeruginosa.

A rapid and simple measurement method for biofilm formation inhibitory activity using 96-pin microtiter plate lids.

The purpose of this study was to develop a rapid and simple measurement method for biofilm formation inhibitory activity, and to screen food additives and foodstuffs that inhibit biofilm formation. The measurement method for biofilm formation inhibitory activity was developed by combining biofilm formation on pins of microtiter plate lids and staining using crystal violet. The optimum conditions for biofilm formation on the pins were established for seven Gram-positive and six Gram-negative bacteria by investigations of media, incubation time, and pin materials. Minimum concentrations of food additives required to inhibit biofilm formation were determined using the proposed method. The values obtained by the proposed and conventional methods agreed well. In addition, by sequential measurements of minimum inhibitory concentrations and minimum bactericidal concentrations using the proposed method, mechanisms of inhibition of biofilm formation were assessed. Furthermore, inhibitory activities of the water extracts of 498 different plant foodstuffs on biofilm formation by Streptococcus mutans were measured; five of the extracts showed potent inhibitory activities. The method proposed here circumvents the tedious and time-consuming conventional method in which biofilms are cultivated on the bottom of wells of microtiter plates.

Biofilm Eradication Activity of Herb and Spice Extracts Alone and in Combination Against Oral and Food-Borne Pathogenic Bacteria.

The purpose of this study was to select herbs and spices with potent biofilm eradication activities. Further, the combined effects of herb and spice extracts against pathogenic biofilms were evaluated. The biofilm eradication activities of ethanol extracts of 104 herbs and spices were measured by combining a colorimetric microbial viability assay with a biofilm formation technique. Ethanol extract of clove had potent biofilm eradication activities against Escherichia coli, Porphyromonas gingivalis, and Streptococcus mutans. Ethanol extracts of eucalyptus and rosemary had potent biofilm eradication activities against P. gingivalis, Staphylococcus aureus and S. mutans. The combination of extracts of clove with eucalyptus or rosemary showed synergistic or additive effects, or both, on biofilm eradication activities. The main biofilm inhibitors in the ethanol extracts of clove, eucalyptus and rosemary were eugenol, macrocarpals and carnosic acid, respectively. The combinations of extracts of clove with eucalyptus or rosemary had potent biofilm eradication activities against oral and food-borne pathogenic bacteria. The findings of the present study reveal that specific combinations of herb and spice extracts may prevent and control biofilm-related oral diseases, food spoilage, and food poisoning.

Prospective single-arm observational study of human chymase inhibitor Polygonum hydropiper L in subjects with hypertension.

Background and Purpose: Human chymase (h-chymase) is a serine protease that forms local angiotensin II and has been proven to be related to onset of hypertension, arteriosclerosis, and post myocardial infarction cardiac remodeling. Since no chymase inhibitor was clinically available, an extensive screening for inhibition of h-chymase in three different extracts (water, hot water,  and ethanol) of approximately 800 food ingredients had been performed and we identified Polygonum hydropiper L (Polygonum). Using a dried and powdered Polygonum, we conducted a prospective, single-arm, pilot study to investigate its safety and antihypertensive effect in subjects with normal high blood pressure to moderate hypertension.Methods: First, a single oral dose of Polygonum powder (4000 mg) was administered to assess acute toxicity. Then, a pilot study was conducted in 11 subjects using the sequence of placebo and Polygonum for 2 weeks each. The dose of Polygonum was increased sequentially (200-2000 mg/day). Home blood pressure and pulse rate were monitored.Results: Oral administration of Polygonum (4000 mg) did not cause any adverse events. In the dose-escalation phase, evening systolic blood pressure was significantly decreased at 800 mg, 2000 mg doses post-treatment (p < 0.05, and p < 0.05, respectively). Depressor responders to Polygonum intake had significantly higher salt intake in spot urine (p < 0.05). No adverse events or reactions occurred.Conclusion: This was the first investigation that an h-chymase inhibitory Polygonum intake for safety and tolerability was proven and, in addition, chymase inhibitory Polygonum appeared to have depressor effect especially in a hypertensive subject with excessive salt intake.

Depressor effect of the young leaves of Polygonum hydropiper Linn. in high-salt induced hypertensive mice.

A novel chymase inhibitor has been reported to have depressor effect in salt-induced hypertension. Therefore, we examined the hypothesis that chymase inhibitory dried young leaves of Polygonum hydropiper (PPH) or young leaves extract of Polygonum hydropiper (PHE) could reduce salt-induced hypertension. In this study, 8-wk old wild-type mice were allocated into three experiments and experiment I included groups, I- normal water drinking, II- high salt (2% NaCl) water (HSW) drinking, and III- HSW plus PPH (500 mg kg-1, orally) for 12-wk. Blood pressure (BP) and heart rate (HR) were measured at baseline and weekly up to wk-12. In experiment II, mice were given HSW for 12-wk followed by 8-wk treatment with PPH plus HSW (62.5, 125, 250 and 500 mg kg-1 for groups I, II, III and IV, respectively). BP and HR were measured at baseline and monthly until wk-12, following weekly for 8-wk. Experiment III comprised of four groups of mice for 12-wk HSW and 8-wk treatment with PHE plus HSW (2.5, 5, 10 and 20 mg kg-1 for groups I-IV, respectively). BP and HR were measured at baseline and monthly up to wk-12, following weekly for 8-wk. Significant reduction in BP and HR were observed in mice treated with PPH (500 mg kg-1) compared to HSW control. PPH reduced BP and HR dose dependently in hypertensive mice and the higher dose showed maximum reduction. PHE at its maximum dose (20 mg kg-1) significantly suppressed BP and HR. Over all, we found that the young leaves of Polygonum hydropiper suppressed salt-induced hypertension.

Single nucleotide polymorphism typing with a surface plasmon resonance-based sensor using hybridization enhancement blockers.

We constructed a simple method for discrimination of single nucleotide polymorphism (SNP) with a surface plasmon resonance (SPR)-based sensor by determining the binding volume (BV) between a target SNP allele and a probe complementary to the target. In the method, a novel additive termed "blocker," which is a short single-stranded DNA complementary to the target, was used. The blocker enhanced the BV of the target only to the full-match probe 10-fold or more, so the SNP alleles could be discriminated readily. The effect of the blocker concentration was also examined. The BV to only a full-match probe increased with increasing the blocker concentration and reached a plateau at the concentration of 300-500 nM. To assess the effectiveness of this method, the SNP associated with progressive rod-cone degeneration in dog was determined. The results of genotyping with the method were in good agreement with those obtained by direct sequencing.

Integration of cytochrome b5 into endoplasmic reticulum membrane: participation of carboxy-terminal portion of the transmembrane domain.

Integration of cytochrome b(5) (b5), a tail-anchored protein located in the endoplasmic reticulum (ER) membrane, into the membrane was studied. Mutation of three amino acids, -Leu-Met-Tyr, at the carboxy-terminal end of the transmembrane segment of b5 to alanines resulted in localization of the mutated protein, b5LMY/AAA, in the cytosol as well as in the ER membrane. When an N-glycosylation site was introduced at the carboxy-terminal end of b5LMY/AAA, a substantial amount of the glycosylated form of the mutant protein was recovered in the cytosol fraction. A portion of the mutant protein recovered in the ER was released from the membrane by incubation with the cytosol fraction, but no further release was observed in the second incubation, suggesting that b5 is present in two different states, loosely-bound and firmly-integrated forms, in the ER membrane. These results suggest that b5 is integrated into the ER membrane via the loosely bound state, in which the carboxy-terminal end of the molecule is inserted into the luminal side of the vesicle but is easily translocated back to the cytosol, and that the three amino acids are important for conversion of the loosely-bound state to the firmly-integrated state.

A critical role for the carboxy terminal region of the proprotein convertase, PACE4A, in the regulation of its autocatalytic activation coupled with secretion.

PACE4A is a member of the mammalian subtilisin-like proprotein convertase family which is responsible for the proteolytic activation of precursors into their biologically active forms. Previously we reported that the maturation of proPACE4A occurs via a intramolecular autoactivation and cleavage of the propeptide is a rate-limiting step for the secretion of PACE4A (Nagahama et al., FEBS Lett. (1998) 434, 155-159). Although PACE4A is a putative secretory enzyme, it matures and is secreted much slower than general secretory proteins. In this study, we investigated the molecular mechanism underlying this slow maturation. The deletion of 25 amino acids at the carboxy terminus is sufficient for a marked acceleration in both the maturation and secretion of PACE4A. The carboxyl-truncated proPACE4A existed only as a monomer-sized form in the endoplasmic reticulum, whereas the wild type of proPACE4A existed in larger forms. Further, the fusion construct of yellow fluorescent protein and the carboxy-terminal sequence of PACE4A associated with the proPACE4A moiety and inhibited maturation. Thus the carboxy terminus of PACE4A functions as a potent autoinhibitor of its activation, resulting in the retention of proPACE4A in the endoplasmic reticulum. These findings indicate that PACE4A activity is highly controlled by a unique system at post-translational level.